Analy&cal Monitoring Considera&ons. Basics of laboratory considera&ons, &ming, and requirements for a business.

Transcription

1 Analy&cal Monitoring Considera&ons Basics of laboratory considera&ons, &ming, and requirements for a business.

2 Refractometry Based on the refrac&ve index of the solu&on A rela&ve measure of the amount of refrac&on occurring as light passes through a glass prism. Higher solute concentra&ons will result in a high angle of refrac&on (difference in transmission). What does this mean for accurate measurements? Solutes other than Sugars will alter the refrac&ve index.

3 Density / Hydrometer Gage the density of a solu&on and compare that to the density of a reference; Water at room temperature. Therefore, solu&ons with a high density (more solute / L solu&on) will have SG > Problems with this? Density (ethanol) = g/ml Other solutes will contribute to changes in density also. Must correct for temperature to be accurate.

4 Digital (electronic) densitometer Based on dampening of oscilla&on through thin- tube capillary. (Anton Paar) Electronic reading made between two points in capillary. Gas bubbles will interfere with reading. In all cases, temperature must be compensated for. In thin- tube, may be harder to compensate for high- temperature liquids.

5 Enzyma&c Determina&on Many assays have been developed based on substrate specific enzyme- mediated reac>ons. Assays are generally >me- sensi>ve, must have stable buffers, and ac>ve enzymes. OYen require very small volume, need micro- pipezors ($) and a Spectrophotometer ($$$) With good sample handling protocols can be very accurate and precise. Can use spectrophotometer for other useful analyses (ethanol, malic acid, ace&c acid, color, phenolics).

6 ph Measure of strength of acid in solu&on. Indirect measure of acid content; but not 1:1 rela&onship. Cri&cal in determining microbial stability. Requirement for proper u&liza&on of Sulfur Dioxide (SO 2 ). Simple measurement provides a lot of valuable informa&on. ph meter (mul>meter) and probe / electrode (s). Need to make sure you maintain calibra&on with fresh buffers.

7 Total Acidity or Titratable Acidity Titra3on of sample against a known base (NaOH; 0.05N or 0.1N) - For apples, assume Malic Acid equivalence. Appreciable amounts of chlorogenic acid, quinic acid, succinc acid. Malic Acid (g/100ml) = V x N x 67 x x v V = volume of NaOH used (ml) N = Normality of NaOH used v = sample volume (ml)

8 Nitrogen In general, we like to know YAN (Yeast Assimilable Nitrogen) leading into fermenta&on. Do we need nitrogen: Diammonium Phosphate (DAP) and Yeast Autolysate. DAP = NH4 Yeast material = Amino Nitrogen, vitamins, minerals Ammonia Ion specific probe Similar to ph probe; determines ion content based on ac&vity of ions in solu&on Ion Specific Probes can be purchased for any number of measurements; NH4 being one. Correlate electric poten&al produced through probe (ions passing through membrane) to concentra&on of that ion.

9 Free Amino Nitrogen (or FAN) NOPA - Based on reac&on of alpha- amino acids (FAN) with orth- pthaldialdehyde (opa). OPA binds to alpha amino acids, resul&ng in an increase in absorbance at 335nm. Proline not an alpha- amino acid, is in higher concentra&ons and not u&lized by yeast during fermenta&on. Prepare a STANDARD CURVE using iso- leucine (known quan&&es). Compare your sample to the standard curve to infer a concentra&on. Amino N + NH4 = YAN Arginine Iso- Leucine Proline

10 Amino Nitrogen (or FAN) Formol Titra3on: Less complicated (instrumenta&on), some issues with chemicals (hygiene and safety). Must work with ven&la&on; formaldehyde is not en&rely safe to work with, must understand risks of chemical exposure. Reac&ng N with formaldehyde, resul&ng in produc&on of an acid. Titra&on of acids with a base (NaOH; similar to TA). - Underes&mates arginine, over- es&mates due to proline Rela&vely simple and effec&ve measure, limited equipment and instrumenta&on. Shively CE, Henick- Kling T. (2001) Comparison of Two Procedures for Assay of Free Amino Nitrogen. Am. J. Enol. Vit. 52:

11 Yeast Popula3on Generally; we want to add a healthy colony of yeast with appropriate nutrients (addi&ons). We want to encourage their rapid growth but not stress them out by deple&ng their O2 and nutrients etc. Shoot for 10 6 cells/ml; rise to 10 8 cell/ml Can count (or es&mate this using a hemacytometer).

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13 - Yeast are typically easy to differen&ate from bacteria, based on size, shape / morphology. - Bacteria can be difficult to differen&ate. - Major organisms are Ace3c acid bacteria (gram nega&ve) and Lac3c acid bacteria (gram posi&ve) - Lysozyme? Gram staining kits are inexpensive, last a long &me, and rela&vely easy to use (4 solu&ons; staining and washing samples).

14 Molecular Form SO 2 Ac&ve form, an&microbial Binds H 2 O 2 (hydrogen peroxide) Inhibits oxida&on through interac&ng with precursors Bisulfite Form HSO 3 - Least effec&ve form; can bind with sugars, phenolics and anthocyanins (bleaching) Will bind acetaldehyde Can reduce browning in white wines; enzyme inhibi&on (PPO) Sulfite Ion Form SO 3 = Can react directly with Oxygen but levels are very low at wine ph

15 How Much Sulfur Do we Need? ph dependent equilibrium Molecular Form SO 2 Bisulfite Form HSO 3 - Sulfite Ion Form SO 3 = Low ph High ph ph meter and this chart

16 Ripper Method: Based on Titra3on of HSO 3 - with Iodine HSO I H 2 0 SO 4 = + 3H + + 3I - Less glassware (flask and bureze only); Iodine reacts with other compounds, (reducing agents; phenolics, aldehydes, ascorbate), and oxygen interferences. 1) Add starch to ~ 50 ml wine (Iodine indicator) Acidify with 5ml Sulfuric acid (25%) Add Sodium bicarbonate (NaHCO 3 ) Titrate against Iodine un&l indicator = blue Calculate FREE SO 2 2) Add 25ml 1M NaOH to 20 ml sample, sit for 10 min. Add starch indicator Acidify with H 2 SO 4 Add NaHCO 3 and &trate with Iodine as before Calculate TOTAL SO 2

17 Analy&cal methods for analysis of SO 2 (Total and Free) Aera3on / oxida3on: Determina&on of Free and Bound SO 2 1) Sample is acidified Air is drawn through the sample; collected into a flask of H 2 O 2 + indicator SO 2 +H = H 2 SO 4 Titrate against NaOH to get total amount of FREE SO 2 2) Same sample is HEATED; liberate BOUND SO 2 Same process of aspira&ng sample into H 2 O 2 Titrated against NaOH to get BOUND SO 2 Total = Free + Bound (mg/l)

18 Ethanol - Hydrometer: Es&ma&on based on loss of sugar (change in gravity from start of fermenta&on). - Inexact but approximate (conversion rate NOT consistent) - Labeled ABV provides enough flexibility to make this adequate for most producers. - Ebulliometers Based on boiling point depression (~$600). - Compare to boiling point of H 2 O at a given &me. - Limita&on of dissolved solids / sugars (prior to sweetening). - Enzyma&c assays: accurate determina&on, less influence of confounding factors. - Requires spectrophotometer and micropipezors - Not simple but ayer some training / trial and error is fine.

19 Vola&le Acidity AceUc Acid Measurement of vola>le acids; those than can be steam dis&lled. Ace3c acid is the major factor; microbial spoilage and legal limits. Load sample in boiling chamber (boiling water bath); Add H 2 O 2. Steam dis&ll sample to capture vola&le acids via condenser. Titrate against base as with TA measurement. (0.05N NaOH).

20 Heat and Cold Stability Can be very simple: Heat samples (e.g. boil for 5 minutes, microwave samples, keep warm (~ F) or Chill (ice bath, fridge, freezer) for a couple days. Look for turbidity / haze forma&on or precipitate. Do you need to cold stabilize, filter, use enzymes (pec&nase- type enzymes).